1. Field of the Invention
This invention relates to cross-linked allophycocyanin, its production and its use in fluorescent assays of various formats.
2. Related Art
Allophycocyanin (APC) is a fluorescent light harvesting protein unique to cyanobacteria and red algae and a member of the phycobiliprotein family of direct fluorescent dyes. APC is excited in the low 600 nm's (650 nm maximum) and emits with a maximum intensity at 660 nm. APC has become more common as a fluorescent label in flow cytometry because of the emergence of multi-laser instrumentation for multi-color detection (e.g., with Helium/Neon laser excitation) and production of tandem dyes for multiplexed assays (Roederer, et al., (1996), Cytometry 24, 191–197). In addition, APC is used as an acceptor from lanthanide dyes for high throughput time resolved fluorescent assays being increasingly used for drug discovery (Park, et al. (1999), Analytical Biochemistry 269, 94–104; Moore, et al. (1999), J Biomol Screening 4, 205–2143; Morris, et al. (1998), British Journal of Haematology 101, 88).
Unfortunately, native APC dissociates into monomers under most assay conditions (e.g., low protein and buffer concentrations). Native APC (herein referred to as “APC”) consists of six phycobiliprotein subunits, made up of three dimers which each contain an alpha and a beta subunit. APC dissociation results in a shift in fluorescence emission from 660 nm and a drop in fluorescence intensity. Dissociation occurs at low concentrations, such as concentrations under 10 μg/mL of APC. Dissociation makes the native APC incompatible for assay conditions commonly used for flow cytometry, microplate assays and high throughput screening (HTS) applications.
Cross-linked and stabilized allophycocyanin preparations (herein referred to as XL-APC) were developed by Glazer and Ong to make this dye more suitable for use in immunoassay (Ong L J & Glazer A N (1985) Physiol Vég 23(1), 777–787). Those authors took a standard preparation of APC and treated it with a chemical cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC), such that an average of one alpha subunit was linked to one beta subunit in a covalent manner in each APC trimer (which contains three of each subunit type). The product was then denatured under 8 M urea to dissociate it into its component parts: alpha and beta monomeric subunits and covalently linked alpha-beta dimeric subunits. The covalently-linked dimers were separated from the monomers using denaturing gel filtration, and then the dimers were placed in an environment that allowed them to reassociate into an (αβ)3 complex that displayed unusually high stability compared to native APC. The resultant material is referred to herein as XL-APC. This material has increased stability in the presence of chaotropic salts (such as sodium perchlorate) or at low concentrations of buffer compared to native APC. When run on a denaturing gel, most of the material runs as a single band that is the covalently linked αβ dimer. In the current art, the higher the percentage of cross-linked αβ dimeric subunits, the better the utility of the XL-APC. A number of XL-APC preparations are commercially available that have various percentages of covalently stabilized αβ dimer in the final product, all greater than 50%.
As seen in FIGS. 1A & 1C, XL-APC has a reduced ration of fluorescence at 650/620 nm versus the native APC. This also is reflected in a reduced ratio of the 650 nm absorbance of APC to the 280 nm absorbance peak reflecting total protein, an indication that the absorbance per molecule has been decreased. One commercially available preparation of XL-APC is called XL665™ (CIS Bio), and this material demonstrates the same decreased ratios of 650/620 fluorescence and 650/280 absorbance observed in the other XL-APC preparations now commercially available.